Acoustic bioreactor processes

ABSTRACT

A series of multi-dimensional acoustic standing waves is set up inside a growth volume of a bioreactor. The acoustic standing waves are used to hold a cell culture in place as a nutrient fluid stream flows through the cell culture. Biomolecules produced by the cell culture are collected by the nutrient fluid stream and separated downstream of the cell culture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/845,531, filed on Jul. 12, 2013. This application is also acontinuation-in-part of U.S. patent application Ser. No. 14/175,766,filed on Feb. 7, 2014, which claimed priority to U.S. Provisional PatentApplication Ser. No. 61/761,717, filed on Feb. 7, 2013. U.S. patentapplication Ser. No. 14/175,766 is also a continuation-in-part of U.S.patent application Ser. No. 14/026,413, filed on Sep. 13, 2013, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/708,641, filed on Oct. 2, 2012, and which is also acontinuation-in-part of U.S. Ser. No. 13/844,754, filed Mar. 15, 2013,which claimed the benefit of U.S. Provisional Patent Application Ser.No. 61/611,159, filed Mar. 15, 2012, and of U.S. Provisional PatentApplication Ser. No. 61/611,240, also filed Mar. 15, 2012, and of U.S.Provisional Patent Application Ser. No. 61/754,792, filed Jan. 21, 2013.These applications are incorporated herein by reference in theirentireties.

BACKGROUND

Growth in the field of biotechnology has been due to many factors, someof which include the improvements in the equipment available forbioreactors. Improvements in equipment have allowed for larger volumesand lower cost for the production of biologically derived materials suchas monoclonal antibodies and recombinant proteins. One of the keycomponents used in the manufacturing processes of new biologically basedpharmaceuticals is the bioreactor and the ancillary processes associatedtherewith.

A modern bioreactor is a very complicated piece of equipment. Itprovides for, among other parameters, the regulation of fluid flowrates, gas content, temperature, pH and oxygen content. All of theseparameters can be tuned to allow the cell culture to be as efficient aspossible of producing the desired biomolecules from the bioreactorprocess. One process for using a bioreactor field is the perfusionprocess. The perfusion process is distinguished from the fed-batchprocess by its lower capital cost and higher throughput.

In the fed-batch process, a culture is seeded in a bioreactor. Thegradual addition of a fresh volume of selected nutrients during thegrowth cycle is used to improve productivity and growth. The product,typically a monoclonal antibody or a recombinant protein, is recoveredafter the culture is harvested. Separating the cells, cell debris andother waste products from the desired product is currently performedusing various types of filters for separation. Such filters areexpensive and become clogged and non-functional as the bioreactormaterial is processed. A fed-batch bioreactor also has high start-upcosts, and generally requires a large volume to obtain a cost-effectiveamount of product at the end of the growth cycle, and such processesinclude large amounts of non-productive downtime.

A perfusion bioreactor processes a continuous supply of fresh media thatis fed into the bioreactor while growth-inhibiting byproducts areconstantly removed. The nonproductive downtime can be reduced oreliminated with a perfusion bioreactor process. The cell densitiesachieved in perfusion culture (30-100 million cells/mL) are typicallyhigher than for fed-batch modes (5-25 million cells/mL). However, aperfusion bioreactor requires a cell retention device to prevent escapeof the culture when byproducts are being removed. These cell retentionsystems add a level of complexity to the perfusion process, requiringmanagement, control, and maintenance for successful operation.Operational issues such as malfunction or failure of the cell retentionequipment has previously been a problem with perfusion bioreactors. Thishas limited their attractiveness in the past.

BRIEF DESCRIPTION

The present disclosure relates, in various embodiments, to systems forproducing biomolecules such as recombinant proteins or monoclonalantibodies, and to processes for separating these desirable productsfrom a cell culture in a bioreactor. Generally, the bioreactor includesa device for producing multi-dimensional standing waves. The standingwaves are used to hold a cell culture in place. A nutrient fluid streamis circulated through the bioreactor past the cell culture to collectbiological products/biomolecules produced by the cell culture. Thebiomolecules can then be separated/harvested from the nutrient fluidstream away from the cell culture.

Disclosed in various embodiments is a system comprising a bioreactor.The bioreactor includes a reaction vessel, an agitator, a feed inlet,and an outlet. A growth volume within the reaction vessel is defined byat least one ultrasonic transducer and a reflector located opposite theat least one ultrasonic transducer. The at least one ultrasonictransducer is driven to produce a multi-dimensional standing wave in thereaction vessel within the growth volume.

Also disclosed herein are processes for collecting biomolecules from acell culture, comprising: suspending the cell culture in a growth volumeof the bioreactor, the bioreactor including at least one ultrasonictransducer and a reflector located opposite the at least one ultrasonictransducer, the at least one ultrasonic transducer being driven toproduce a multi-dimensional acoustic standing wave that holds the cellculture in the growth volume; and flowing a nutrient fluid streamthrough the cell culture to collect the biomolecules.

The bioreactor may further comprise a secondary filtering system locatedbetween the growth volume and a bioreactor outlet. The secondaryfiltering system is activated if the multi-dimensional acoustic standingwave fails. The multi-dimensional acoustic standing wave is generallyoperated in resonance.

The bioreactor may have an array of elements that form the ultrasonictransducer. Alternatively or in addition, each ultrasonic transducer mayproduce a plurality of multi-dimensional acoustic standing waves.

In particular embodiments, the bioreactor does not include an impeller(i.e a physical agitator) within the growth volume. The cell culture is,in particular embodiments, composed of Chinese hamster ovary (CHO)cells. The biomolecules produced thereby can be monoclonal antibodies orrecombinant proteins.

The multi-dimensional acoustic standing wave may have an axial forcecomponent and a lateral force component which are of the same order ofmagnitude. The bioreactor can be operated as a perfusion bioreactor. Thebioreactor can include a jacket that is used to regulate the temperatureof the fluid in the growth volume.

In particular embodiments, the ultrasonic transducer comprises apiezoelectric material that can vibrate in a higher order mode shape.The piezoelectric material may have a square or rectangular shape.

The ultrasonic transducer may comprise: a housing having a top end, abottom end, and an interior volume; and a crystal at the bottom end ofthe housing having an exposed exterior surface and an interior surface,the crystal being able to generate acoustic waves when driven by avoltage signal. In some embodiments, a backing layer contacts theinterior surface of the crystal, the backing layer being made of asubstantially acoustically transparent material. The substantiallyacoustically transparent material can be balsa wood, cork, or foam. Thesubstantially acoustically transparent material may have a thickness ofup to 1 inch. The substantially acoustically transparent material can bein the form of a lattice. In other embodiments, an exterior surface ofthe crystal is covered by a wear surface material with a thickness of ahalf wavelength or less, the wear surface material being a urethane,epoxy, or silicone coating. The exterior surface of the crystal may alsohave wear surface formed from a matching layer or wear plate of materialadhered to the exterior surface of the crystal. The matching layer orwear plate may be composed of aluminum oxide. In yet other embodiments,the crystal has no backing layer or wear layer.

The multi-dimensional acoustic standing wave can be a three-dimensionalstanding wave. The reflector and/or the ultrasonic transducer may have anon-planar surface.

These and other non-limiting characteristics are more particularlydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 illustrates a single standing acoustic wave generated by anultrasonic transducer and a reflector.

FIG. 2 is an illustration comparing a conventional fed-batch bioreactorsystem with a perfusion bioreactor system.

FIG. 3 is a cross-sectional view that shows the various components of abioreactor of the present disclosure.

FIG. 4 is a cut-out view of a tubular bioreactor and the growth volumetherein. A plurality of ultrasonic transducers is used to generatestanding waves that hold a cell culture in place. Arrows illustrate anupward flow of a nutrient fluid stream through the standing waves andthe cell culture held therein.

FIG. 5 is a cross-sectional diagram of a conventional ultrasonictransducer.

FIG. 6 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and nobacking layer or wear plate is present.

FIG. 7 is a cross-sectional diagram of an ultrasonic transducer of thepresent disclosure. An air gap is present within the transducer, and abacking layer and wear plate are present.

FIG. 8 is an illustration of a piezoelectric array that can be used toproduce multi-dimensional standing waves.

FIG. 9 is a graph of electrical impedance amplitude versus frequency fora square transducer driven at different frequencies.

FIG. 10 illustrates the trapping line configurations for seven of thepeak amplitudes of FIG. 9 from the direction orthogonal to fluid flow.

FIG. 11 is a computer simulation of the acoustic pressure amplitude(right-hand scale in Pa) and transducer out of plane displacement(left-hand scale in meters). The text at the top of the left-hand scalereads “×10⁻⁷”. The text at the top of the left-hand scale by theupward-pointing triangle reads “1.473×10⁻⁶”. The text at the bottom ofthe left-hand scale by the downward-pointing triangle reads“1.4612×10⁻¹⁰”. The text at the top of the right-hand scale reads“×10⁶”. The text at the top of the right-hand scale by theupward-pointing triangle reads “1.1129×10⁶”. The text at the bottom ofthe right-hand scale by the downward-pointing triangle reads “7.357”.The triangles show the maximum and minimum values depicted in thisfigure for the given scale. The horizontal axis is the location withinthe chamber along the X-axis, in inches, and the vertical axis is thelocation within the chamber along the Y-axis, in inches.

FIG. 12 shows the In-Plane and Out-of-Plane displacement of a crystalwhere composite waves are present.

FIG. 13 shows an exploded view of a bioreactor that can be used, havingone growth volume.

FIG. 14 shows an exploded view of a bioreactor having two stacked growthvolumes.

DETAILED DESCRIPTION

The present disclosure may be understood more readily by reference tothe following detailed description of desired embodiments and theexamples included therein. In the following specification and the claimswhich follow, reference will be made to a number of terms which shall bedefined to have the following meanings.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

The term “comprising” is used herein as requiring the presence of thenamed component and allowing the presence of other components. The term“comprising” should be construed to include the term “consisting of”,which allows the presence of only the named component, along with anyimpurities that might result from the manufacture of the namedcomponent.

Numerical values should be understood to include numerical values whichare the same when reduced to the same number of significant figures andnumerical values which differ from the stated value by less than theexperimental error of conventional measurement technique of the typedescribed in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint andindependently combinable (for example, the range of “from 2 grams to 10grams” is inclusive of the endpoints, 2 grams and 10 grams, and all theintermediate values). The endpoints of the ranges and any valuesdisclosed herein are not limited to the precise range or value; they aresufficiently imprecise to include values approximating these rangesand/or values.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (forexample, it includes at least the degree of error associated with themeasurement of the particular quantity). When used in the context of arange, the modifier “about” should also be considered as disclosing therange defined by the absolute values of the two endpoints. For example,the range of “from about 2 to about 10” also discloses the range “from 2to 10.”

It should be noted that many of the terms used herein are relativeterms. For example, the terms “upper” and “lower” are relative to eachother in location, i.e. an upper component is located at a higherelevation than a lower component in a given orientation, but these termscan change if the device is flipped. The terms “inlet” and “outlet” arerelative to a fluid flowing through them with respect to a givenstructure, e.g. a fluid flows through the inlet into the structure andflows through the outlet out of the structure. The terms “upstream” and“downstream” are relative to the direction in which a fluid flowsthrough various components, i.e. the flow fluids through an upstreamcomponent prior to flowing through the downstream component. It shouldbe noted that in a loop, a first component can be described as beingboth upstream of and downstream of a second component.

The terms “horizontal” and “vertical” are used to indicate directionrelative to an absolute reference, i.e. ground level. However, theseterms should not be construed to require structures to be absolutelyparallel or absolutely perpendicular to each other. For example, a firstvertical structure and a second vertical structure are not necessarilyparallel to each other. The terms “upwards” and “downwards” are alsorelative to an absolute reference; an upwards flow is always against thegravity of the earth.

The present application refers to “the same order of magnitude.” Twonumbers are of the same order of magnitude if the quotient of the largernumber divided by the smaller number is a value less than 10.

The term “agitator” is used herein to refer to any device or systemwhich can be used to cause mixing of a fluid volume, such that materialin the fluid volume is dispersed and becomes more homogeneous. The term“impeller” is used to refer to a physical agitator, such as a blade.Examples of agitators which are not impellers may include aerators(which use air).

Bioreactors are useful for making biomolecules such as recombinantproteins or monoclonal antibodies. Very generally, cells are cultured ina bioreactor vessel with media in order to produce the desired product,and the desired product is then harvested by separation from the cellsand media. The use of mammalian cell cultures including Chinese hamsterovary (CHO), NS0 hybridoma cells, baby hamster kidney (BHK) cells, andhuman cells has proven to be a very efficacious way ofproducing/expressing the recombinant proteins and monoclonal antibodiesrequired of today's pharmaceuticals.

Two general types of bioreactor processes exist: fed-batch andperfusion. Many factors favor the use of a perfusion bioreactor process.The capital and start-up costs for perfusion bioreactors are lower,smaller upstream and downstream capacity is required, and the processuses smaller volumes and fewer seed steps than fed-batch methods. Aperfusion bioreactor process also lends itself better to development,scale-up, optimization, parameter sensitivity studies, and validation.

Recent developments in perfusion bioreactor technology also favor itsuse. Control technology and general support equipment is improving forperfusion bioreactors, increasing the robustness of perfusion processes.The perfusion process can now be scaled up to bioreactors having avolume up to 1000 liters (L). Better cell retention systems forperfusion bioreactors result in lower cell loss and greater celldensities than have been seen previously. Cell densities greater than 50million cells/mL are now achievable, compared to fed-batch celldensities of around 20 million cells/mL. Lower contamination andinfection rates have improved the output of perfusion bioreactors.Higher product concentrations in the harvest and better yields withoutsignificant increase in cost have thus resulted for perfusion processes.

There is a need for improved filtration processes in a fed-batchbioreactor process. There is also a need in perfusion bioreactorprocesses for improving retention of cells in the bioreactor while thebiomolecules are continuously harvested.

Briefly, the present disclosure relates to the generation ofthree-dimensional (3-D) or multi-dimensional acoustic standing wavesfrom one or more piezoelectric transducers, where the transducers areelectrically or mechanically excited such that they move in amulti-excitation mode. The types of waves thus generated can becharacterized as composite waves, with displacement profiles that aresimilar to leaky symmetric (also referred to as compressional orextensional) Lamb waves. The waves are leaky because they radiate intothe water layer, which result in the generation of the acoustic standingwaves in the water layer. Symmetric Lamb waves have displacementprofiles that are symmetric with respect to the neutral axis of thepiezoelectric element, which causes multiple standing waves to begenerated in a 3-D space. Through this manner of wave generation, ahigher lateral trapping force is generated than if the piezoelectrictransducer is excited in a “piston” mode where only a single, planarstanding wave is generated. Thus, with the same input power to apiezoelectric transducer, the 3-D or multi-dimensional acoustic standingwaves can have a higher lateral trapping force which may be up to andbeyond 10 times stronger than a single acoustic standing wave generatedin piston mode. This can be used to hold the cell culture within adefined volume (referred to herein as a “growth volume”) while the fluidcontents and desired byproducts are removed.

Acoustophoresis is a low-power, no-pressure-drop, no-clog, solid-stateapproach to separate solids from fluids, i.e. it is used to achieveseparations that are more typically performed with porous filters, butit has none of the disadvantages of filters. In particular, the presentdisclosure provides bioreactors that operate at the macro-scale toseparate cell cultures in flowing systems with high flow rates. Thebioreactor uses a high intensity three dimensional ultrasonic standingwave that results in an acoustic radiation force that is larger than thecombined effects of fluid drag and buoyancy or gravity, and is thereforeable to trap (i.e., hold in place) a suspended phase (i.e. cells andcell cultures) in the standing wave. The retained cells may clump oragglomerate. A nutrient fluid stream can then be flowed/circulatedthrough the cell culture to provide nutrition and oxygenation to thecells, and to capture the biomolecules produced by the cells. Thestanding waves are also believed to stimulate the cell culture, so as toincrease the rate of expression of the desired biomolecules. Thisprovides a self-contained “acoustic bioreactor” where the biomoleculesmay be continuously harvested, and at an accelerated rate, from the cellculture. The present systems have the ability to create acousticultrasonic standing wave fields that can hold the cell cultures in placein a flow field with a linear velocity ranging from 0.1 mm/sec tovelocities exceeding 1 cm/s.

The multi-dimensional ultrasonic acoustic standing waves can be used totrap, i.e., hold stationary, a cell culture in a fluid stream (e.g. cellculture medium or nutrient fluid stream). The scattering of the acousticfield off the cells results in a three dimensional acoustic radiationforce, which acts as a three-dimensional trapping field. The acousticradiation force is proportional to the particle volume (e.g. the cube ofthe radius) when the particle is small relative to the wavelength. It isproportional to frequency and the acoustic contrast factor. It alsoscales with acoustic energy (e.g. the square of the acoustic pressureamplitude). For harmonic excitation, the sinusoidal spatial variation ofthe force is what drives the cells to the stable positions within thestanding waves. When the acoustic radiation force exerted on the cell isstronger than the combined effect of fluid drag force andbuoyancy/gravitational force, the cell is trapped within the acousticstanding wave field. The action of the acoustic forces on the trappedcells results in concentration, agglomeration and/or coalescence.

Generally, the 3-D or multi-dimensional acoustic standing wave(s) isoperated at a voltage and frequency such that the biomolecule-producingcell culture, such as Chinese hamster ovary cells (CHO cells), the mostcommon host for the industrial production of recombinant proteintherapeutics, are held in place by the ultrasonic standing wave, i.e.,remain in a stationary position. Within each nodal plane, the CHO cellsare trapped in the minima of the acoustic radiation potential. Most celltypes present a higher density and lower compressibility than the mediumin which they are suspended, so that the acoustic contrast factorbetween the cells and the medium has a positive value. As a result, theaxial acoustic radiation force (ARF) drives the CHO cells towards thestanding wave pressure nodes. The axial component of the acousticradiation force drives the cells, with a positive contrast factor, tothe pressure nodal planes, whereas cells or other particles with anegative contrast factor are driven to the pressure anti-nodal planes.The radial or lateral component of the acoustic radiation force helpstrap the cells. The radial or lateral component of the ARF is largerthan the combined effect of fluid drag force and gravitational force.For small cells or emulsions the drag force F_(D) can be expressed as:

${{\overset{->}{F}}_{D} = {4{\pi\mu}_{f}{{R_{p}\left( {{\overset{->}{U}}_{f} - {\overset{->}{U}}_{p}} \right)}\left\lbrack \frac{1 + {\frac{3}{2}\hat{\mu}}}{1 + \hat{\mu}} \right\rbrack}}},$

where U_(f) and U_(p) are the fluid and cell velocity, R_(p) is theparticle radius, μ_(f) and μ_(p) are the dynamic viscosity of the fluidand the cells, and {circumflex over (μ)}=μ_(p)/μ_(f) is the ratio ofdynamic viscosities. The buoyancy force F_(B) is expressed as:

F _(B)=4/3πR _(p) ³(ρ_(f)−ρ_(p)).

For a cell to be trapped in the ultrasonic standing wave, the forcebalance on the cell must be zero, and therefore an expression forlateral acoustic radiation force F_(LRF) can be found, which is givenby:

F _(LRF) =F _(D) +F _(B).

For a cell of known size and material property, and for a given flowrate, this equation can be used to estimate the magnitude of the lateralacoustic radiation force.

The theoretical model that is used to calculate the acoustic radiationforce is based on the formulation developed by Gor′kov.¹⁷ The primaryacoustic radiation force F_(A) is defined as a function of a fieldpotential U, F_(A)=−∇(U), where the field potential U is defined as

${U = {V_{0}\left\lbrack {{\frac{\langle p^{2}\rangle}{2\rho_{f}c_{f}^{2}}f_{1}} - {\frac{3\rho_{f}{\langle u^{2}\rangle}}{4}f_{2}}} \right\rbrack}},$

and f₁ and f₂ are the monopole and dipole contributions defined by

${f_{1} = {1 - \frac{1}{{\Lambda\sigma}^{2}}}},{f_{2} = \frac{2\left( {\Lambda - 1} \right)}{{2\Lambda} + 1}},$

where p is the acoustic pressure, u is the fluid particle velocity,

is the ratio of cell density ρ_(p) to fluid density ρ_(f), σ is theratio of cell sound speed c_(p) to fluid sound speed c_(f),

_(o) is the volume of the cell, and < > indicates time averaging overthe period of the wave.

The lateral force of the total acoustic radiation force (ARF) generatedby the ultrasonic transducers of the present disclosure is significantand is sufficient to overcome the fluid drag force at linear velocitiesof up to 1 cm/s and beyond. This lateral ARF can thus be used to retaincells in a particular volume of a bioreactor while the bioreactorprocess continues. This is especially true for a perfusion bioreactor.

In a perfusion bioreactor system, it is desirable to be able to filterand separate the cells and cell debris from the expressed materials thatare in the fluid stream (i.e. cell culture media or nutrient fluidstream). The expressed materials are composed of biomolecules such asrecombinant proteins or monoclonal antibodies, and are the desiredproduct to be recovered.

The standing waves can be used to trap the cells and cell debris presentin the cell culture media. The cells, having a positive contrast factor,move to the nodes (as opposed to the anti-nodes) of the standing wave.As the cells agglomerate at the nodes of the standing wave, there isalso a physical scrubbing of the cell culture media that occurs wherebymore cells are trapped as they come in contact with the cells that arealready held within the standing wave. This generally separates thecells from the cell culture media. The expressed biomolecules remain inthe nutrient fluid stream (i.e. cell culture medium).

Desirably, the ultrasonic transducer(s) generate a three-dimensional ormulti-dimensional acoustic standing wave in the fluid that exerts alateral force on the suspended particles to accompany the axial force soas to increase the particle trapping capabilities of the standing wave.Typical results published in literature state that the lateral force istwo orders of magnitude smaller than the axial force. In contrast, thetechnology disclosed in this application provides for a lateral force tobe of the same order of magnitude as the axial force.

The bioreactors of the present disclosure are designed to maintain ahigh intensity multi-dimensional acoustic standing wave. The device isdriven by a function generator and amplifier (not shown). The deviceperformance is monitored and controlled by a computer. It may benecessary, at times, due to acoustic streaming, to modulate thefrequency or voltage amplitude of the standing wave. This may be done byamplitude modulation and/or by frequency modulation.

FIG. 1 illustrates a single standing wave system 100 that is comprisedof a reflector plate 101 and an ultrasonic transducer 103 that is set toresonate so as to form a standing wave 102. Excitation frequenciestypically in the range from hundreds of kHz to tens of MHz are appliedby the transducer 103. One or more standing waves are created betweenthe transducer 103 and the reflector 101. The standing wave is the sumof two propagating waves that are equal in frequency and intensity andthat are traveling in opposite directions, i.e. from the transducer tothe reflector and back. The propagating waves destructively interferewith each other and thus generate the standing wave. A dotted line 105is used to indicate the amplitude. A node is a point where the wave hasminimum amplitude, and is indicated with reference numeral 107. Ananti-node is a point where the wave has maximum amplitude, and isindicated with reference numeral 109.

FIG. 2 is a schematic diagram that compares a conventional fed-batchbioreactor system 201 (left side) with a conventional perfusionbioreactor system 202 (right side). Beginning with the fed-batchbioreactor on the left, the bioreactor 210 includes a reaction vessel220. The cell culture media is fed to the reaction vessel through a feedinlet 222. An agitator 225 is used to circulate the media throughout thecell culture. Here, the agitator is depicted as a set of rotatingblades, though any type of system that causes circulation iscontemplated. The bioreactor permits growth of a seed culture through agrowth/production cycle, during which time debris, waste and unusablecells will accumulate in the bioreactor and the desired product (e.g.biomolecules such as monoclonal antibodies, recombinant proteins,hormones, etc.) will be produced as well. Due to this accumulation, thereaction vessel of a fed-batch process is typically much larger thanthat in a perfusion process. The desired product is then harvested atthe end of the production cycle. The reaction vessel 220 also includesan outlet 224 for removing material.

Turning now to the perfusion bioreactor 202 on the right-hand side,again, the bioreactor includes a reaction vessel 220 with a feed inlet222 for the cell culture media. An agitator 225 is used to circulate themedia throughout the cell culture. An outlet 224 of the reaction vesselis fluidly connected to the inlet 232 of a filtering device 230, andcontinuously feeds the media (containing cells and desired product) to afiltering device. The filtering device is located downstream of thereaction vessel, and separates the desired product from the cells. Thefiltering device 230 has two separate outlets, a product outlet 234 anda recycle outlet 236. The product outlet 234 fluidly connects thefiltering device 230 to a containment vessel 240 downstream of thefiltering device, which receives a concentrated flow of the desiredproduct (plus media) from the filtering device. From there, furtherprocessing/purification can occur to isolate/recover the desiredproduct. The recycle outlet 236 fluidly connects the filtering device230 back to a recycle inlet 226 of the reaction vessel 220, and is usedto send the cells and cell culture media back into the reaction vesselfor continued growth/production. Put another way, there is a fluid loopbetween the reaction vessel and the filtering device. The reactionvessel 220 in the perfusion bioreactor system 202 has a continuousthroughput of product and thus can be made smaller. The filteringprocess is critical to the throughput of the perfusion bioreactor. Apoor filtering process will allow for only low throughput and result inlow yields of the desired product.

FIG. 3 is a cross-sectional view of a bioreactor 300 used in the systemsof the present disclosure. As illustrated here, the bioreactor includesa reaction vessel 320 having an internal volume 323. A feed inlet 322 atthe top of the vessel is used to feed a nutrient fluid stream into thevessel, and can also be used to feed in additional cells for maintainingthe cell culture. An agitator 325 is present in the form of an impellerblade. An outlet 324 is shown at the bottom of the vessel. An aerator(not shown) can be used to provide gas to the internal volume. Sensors314 are shown at the top right of the vessel. A pump 316 is illustratedfor feeding the nutrient fluid stream into the vessel, as is anotherpump 318 for removing the nutrient fluid stream from the vessel. Thesepumps are used to circulate the nutrient fluid stream through the cellculture and furnish nutrition and oxygenation to keep the cells viableand productive. The pumps also deliver the produced biomolecules toanother portion of the bioreactor (not shown) where these biomoleculescan be further filtered and separated downstream of the reaction vessel.

The reaction vessel also includes an ultrasonic transducer 330 on oneside of the vessel, and a reflector 332 located on another side oppositethe ultrasonic transducer. A growth volume 334 is present between thetransducer and the reflector (illustrated with dotted lines). Amulti-dimensional standing wave (not shown) is generated between thetransducer and the reflector that holds the cell culture in the growthvolume. It is noted that the growth volume 334 is a portion of theinternal volume 323. It is also noted that the blade of the agitator 325is not located within the growth volume 334, because its presence candisrupt the standing wave

A thermal jacket 310 surrounds the reaction vessel, and is used toregulate the temperature of the internal volume 323 and the cellculture. In this regard, it is usually desirable to maintain thetemperature of the cell culture below 38° C. to prevent compromise ofthe cells. The thermal jacket is usually a chilling system used tomitigate any excess heat generated by the ultrasonic transducers. It isnoted that the thermal jacket typically contains atemperature-regulating fluid. The standing wave created by thetransducer 330 and reflector 332 can propagate through the jacket andthe temperature-regulating fluid therein, and still continue to operatein the reaction vessel to hold the cell culture in place.

A secondary filtering system 312 is located between the growth volume334 and the outlet 324. It is contemplated that in the event thestanding waves fail to hold the cell culture in place, the secondaryfiltering system will operate to keep the cell culture within thereaction vessel and maintain their separation from the producedbiomolecules. This could occur, for example, if a high percentage of theultrasonic transducers fail, or if resonance is lost, or if the power iscut off to the reaction vessel.

During operation, the nutrient fluid stream is added into the reactionvessel through the feed inlet 322. The contents of the reaction vesselare mixed with the agitator 325. The desired product (e.g. biomolecules)is continuously produced by cells located within the growth volume 334,and are separated from the cell culture by the nutrient fluid steamflowing through the growth volume. The nutrient fluid stream, containingthe biomolecular product, is drawn from the reaction vessel throughoutlet 324. From there, the nutrient fluid stream can be processed toisolate the desired product.

After processing, any cells and the nutrient fluid can be recycled backto the reaction vessel. In this regard, the present disclosure shouldnot be construed as stating that no cells ever escape the standing waveand the growth volume.

It is noted that in FIG. 3, the reaction vessel inlet 322 is depicted atthe top of the vessel and the outlet 324 is depicted at the bottom ofthe vessel. This arrangement can be reversed if desired, for exampledepending on the desired product to be obtained.

FIG. 4 is another illustration of the reaction vessel 420 of abioreactor. Here, the reaction vessel is tubular, with the outlet at thetop and the inlet at the bottom of the vessel, with fluid flow beingindicated by arrows 405. An array of transducers 430 is arrangedvertically on one side, and an array of reflectors 432 is arranged on anopposite side from the transducers. Waves 401 are transmitted from thetransducer to the reflector, and waves 402 bounce back from thereflector to the transducer. The cell culture is held in place at thenodes of the standing wave thus generated, and is indicated withreference number 403.

The reaction vessel may be tubular, cubic, or another polygonal shape.The flow of the nutrient fluid stream through the reaction vessel of thebioreactor may be vertical, horizontal, or any angle in between. Thecombination of the ultrasonic transducers and the reflectors set up theresonant waves in the interior of the reaction vessel. The standingwaves hold the cell culture at their net zero pressure nodes. Theultrasonic transducers and reflectors may be set perpendicular or atanother angle to the fluid flow of the nutrient fluid stream through theacoustic bioreactor. This will allow for greater holding strength of theacoustic standing wave for the cell culture. The reflector may be apassive shape that is flat or is non-planar, or alternatively may itselfbe an active piezoelectric element that can change its shape to maintainresonance, but cannot itself generate an acoustic wave.

It may be helpful now to describe the ultrasonic transducer(s) used inthe acoustophoretic filtering device in more detail. FIG. 5 is across-sectional diagram of a conventional ultrasonic transducer. Thistransducer has a wear plate 50 at a bottom end, epoxy layer 52, ceramiccrystal 54 (made of, e.g. Lead Zirconate Titanate (PZT)), an epoxy layer56, and a backing layer 58. On either side of the ceramic crystal, thereis an electrode: a positive electrode 61 and a negative electrode 63.The epoxy layer 56 attaches backing layer 58 to the crystal 54. Theentire assembly is contained in a housing 60 which may be made out of,for example, aluminum. An electrical adapter 62 provides connection forwires to pass through the housing and connect to leads (not shown) whichattach to the crystal 54. Typically, backing layers are designed to adddamping and to create a broadband transducer with uniform displacementacross a wide range of frequency and are designed to suppress excitationat particular vibrational eigen-modes. Wear plates are usually designedas impedance transformers to better match the characteristic impedanceof the medium into which the transducer radiates. However, theoscillating pressure and heating of the crystal can cause the wear plateto separate from the crystal.

FIG. 8 is a cross-sectional view of an ultrasonic transducer 81 of thepresent disclosure, which is used in the acoustophoretic filteringdevices of the present disclosure. Transducer 81 has an aluminum housing82. A PZT crystal 86 defines the bottom end of the transducer, and isexposed from the exterior of the housing. The crystal is supported onits perimeter by a small elastic layer 98, e.g. silicone or similarmaterial, located between the crystal and the housing. Put another way,no wear layer is present. The housing may also be composed of a moreelectrically conductive material, such as steel. The housing may also begrounded to the negative side of the transducer.

Screws (not shown) attach an aluminum top plate 82 a of the housing tothe body 82 b of the housing via threads 88. The top plate includes aconnector 84 to pass power to the PZT crystal 86. The bottom and topsurfaces of the PZT crystal 86 are each connected to an electrode(positive and negative), such as silver or nickel. A wrap-aroundelectrode tab 90 connects to the bottom electrode and is isolated fromthe top electrode. Electrical power is provided to the PZT crystal 86through the electrodes on the crystal, with the wrap-around tab 90 beingthe ground connection point. Note that the crystal 86 has no backinglayer or epoxy layer as is present in FIG. 5. Put another way, there isan air gap 87 in the transducer between aluminum top plate 82 a and thecrystal 86 (i.e. the air gap is completely empty). A minimal backing 58and/or wear plate 50 may be provided in some embodiments, as seen inFIG. 9.

The transducer design can affect performance of the system. A typicaltransducer is a layered structure with the ceramic crystal bonded to abacking layer and a wear plate. Because the transducer is loaded withthe high mechanical impedance presented by the standing wave, thetraditional design guidelines for wear plates, e.g., half wavelengththickness for standing wave applications or quarter wavelength thicknessfor radiation applications, and manufacturing methods may not beappropriate. Rather, in one embodiment of the present disclosure thetransducers, there is no wear plate or backing, allowing the crystal tovibrate in one of its eigenmodes with a high Q-factor. The vibratingceramic crystal/disk is directly exposed to the fluid flowing throughthe flow chamber.

Removing the backing (e.g. making the crystal air backed) also permitsthe ceramic crystal to vibrate at higher order modes of vibration withlittle damping (e.g. higher order modal displacement). In a transducerhaving a crystal with a backing, the crystal vibrates with a moreuniform displacement, like a piston. Removing the backing allows thecrystal to vibrate in a non-uniform displacement mode. The higher orderthe mode shape of the crystal, the more nodal lines the crystal has. Thehigher order modal displacement of the crystal creates more trappinglines, although the correlation of trapping line to node is notnecessarily one to one, and driving the crystal at a higher frequencywill not necessarily produce more trapping lines.

In some embodiments, the crystal may have a backing that minimallyaffects the Q-factor of the crystal (e.g. less than 5%). The backing maybe made of a substantially acoustically transparent material such asbalsa wood, foam, or cork which allows the crystal to vibrate in ahigher order mode shape and maintains a high Q-factor while stillproviding some mechanical support for the crystal. The backing layer maybe a solid, or may be a lattice having holes through the layer, suchthat the lattice follows the nodes of the vibrating crystal in aparticular higher order vibration mode, providing support at nodelocations while allowing the rest of the crystal to vibrate freely. Thegoal of the lattice work or acoustically transparent material is toprovide support without lowering the Q-factor of the crystal orinterfering with the excitation of a particular mode shape.

Placing the crystal in direct contact with the fluid also contributes tothe high Q-factor by avoiding the dampening and energy absorptioneffects of the epoxy layer and the wear plate. Other embodiments mayhave wear plates or a wear surface to prevent the PZT, which containslead, contacting the host fluid. This may be desirable in, for example,biological applications such as separating blood. Such applicationsmight use a wear layer such as chrome, electrolytic nickel, orelectroless nickel. Chemical vapor deposition could also be used toapply a layer of poly(p-xylylene) (e.g. Parylene) or other polymer.Organic and biocompatible coatings such as silicone or polyurethane arealso usable as a wear surface.

In some embodiments, the ultrasonic transducer has a 1 inch diameter anda nominal 2 MHz resonance frequency. Each transducer can consume about28 W of power for droplet trapping at a flow rate of 3 GPM. Thistranslates to an energy cost of 0.25 kW hr/m³. This is an indication ofthe very low cost of energy of this technology. Desirably, eachtransducer is powered and controlled by its own amplifier. In otherembodiments, the ultrasonic transducer uses a square crystal, forexample with 1″×1″ dimensions. Alternatively, the ultrasonic transducercan use a rectangular crystal, for example with 1″×2.5″ dimensions.Power dissipation per transducer was 10 W per 1″×1″ transducercross-sectional area and per inch of acoustic standing wave span inorder to get sufficient acoustic trapping forces. For a 4″ span of anintermediate scale system, each 1″×1″ square transducer consumes 40 W.The larger 1″×2.5″ rectangular transducer uses 100 W in an intermediatescale system. The array of three 1″×1″ square transducers would consumea total of 120 W and the array of two 1″×2.5″ transducers would consumeabout 200 W. Arrays of closely spaced transducers represent alternatepotential embodiments of the technology. Transducer size, shape, number,and location can be varied as desired to generate desiredthree-dimensional acoustic standing wave patterns.

FIG. 8 is an illustration of a piezoelectric ultrasonic transducer 800that may also be utilized to generate multiple standing waves. The base801 of the transducer has an array formed from multiple piezoelectricelements 802 on the surface. These piezoelectric elements may be formedon the surface in a variety of ways, including adhesion of piezoelectriccrystals, photomasking and deposition techniques such as those utilizedin the electronic industry. For example, the surface of a piezoelectriccrystal can be cut in a pattern to a certain depth, and the cut-awayareas are then filled with a secondary material to isolate theindividual areas to form the resulting pattern on the piezoelectriccrystal surface.

The size, shape, and thickness of the transducer determine thetransducer displacement at different frequencies of excitation, which inturn affects separation efficiency. Typically, the transducer isoperated at frequencies near the thickness resonance frequency (halfwavelength). Gradients in transducer displacement typically result inmore trapping locations for the cells/biomolecules. Higher order modaldisplacements generate three-dimensional acoustic standing waves withstrong gradients in the acoustic field in all directions, therebycreating equally strong acoustic radiation forces in all directions,leading to multiple trapping lines, where the number of trapping linescorrelate with the particular mode shape of the transducer.

To investigate the effect of the transducer displacement profile onacoustic trapping force and separation efficiencies, an experiment wasrepeated ten times using a 1″×1″ square transducer, with all conditionsidentical except for the excitation frequency. Ten consecutive acousticresonance frequencies, indicated by circled numbers 1-9 and letter A onFIG. 9, were used as excitation frequencies. The conditions wereexperiment duration of 30 min, a 1000 ppm oil concentration ofapproximately 5-micron SAE-30 oil droplets, a flow rate of 500 ml/min,and an applied power of 20 W. Oil droplets were used because oil isdenser than water, and can be separated from water usingacoustophoresis.

FIG. 9 shows the measured electrical impedance amplitude of a squaretransducer as a function of frequency in the vicinity of the 2.2 MHztransducer resonance. The minima in the transducer electrical impedancecorrespond to acoustic resonances of the water column and representpotential frequencies for operation. Numerical modeling has indicatedthat the transducer displacement profile varies significantly at theseacoustic resonance frequencies, and thereby directly affects theacoustic standing wave and resulting trapping force. Since thetransducer operates near its thickness resonance, the displacements ofthe electrode surfaces are essentially out of phase. The typicaldisplacement of the transducer electrodes is not uniform and variesdepending on frequency of excitation. As an example, at one frequency ofexcitation with a single line of trapped oil droplets, the displacementhas a single maximum in the middle of the electrode and minima near thetransducer edges. At another excitation frequency, the transducerprofile has multiple maxima leading to multiple trapped lines of oildroplets. Higher order transducer displacement patterns result in highertrapping forces and multiple stable trapping lines for the captured oildroplets.

As the oil-water emulsion passed by the transducer, the trapping linesof oil droplets were observed and characterized. The characterizationinvolved the observation and pattern of the number of trapping linesacross the fluid channel, as shown in FIG. 10, for seven of the tenresonance frequencies identified in FIG. 9. Different displacementprofiles of the transducer can produce different (more) trapping linesin the standing waves, with more gradients in displacement profilegenerally creating higher trapping forces and more trapping lines.

FIG. 11 is a numerical model showing a pressure field that matches the 9trapping line pattern. The numerical model is a two-dimensional model;and therefore only three trapping lines are observed. Two more sets ofthree trapping lines exist in the third dimension perpendicular to theplane of the page.

In the present systems, the system is operated at a voltage andfrequency such that the cells (that make up the cell culture) aretrapped by the ultrasonic standing wave, i.e., remain in a stationaryposition. The cells are collected along well-defined trapping lines,separated by half a wavelength. Within each nodal plane, the cells aretrapped in the minima of the acoustic radiation potential. The axialcomponent of the acoustic radiation force drives cells with a positivecontrast factor to the pressure nodal planes, whereas cells with anegative contrast factor are driven to the pressure anti-nodal planes.The radial or lateral component of the acoustic radiation force is theforce that traps the cell. It therefore must be larger than the combinedeffect of fluid drag force and gravitational force. In systems usingtypical transducers, the radial or lateral component of the acousticradiation force is typically several orders of magnitude smaller thanthe axial component of the acoustic radiation force. However, thelateral force generated by the transducers of the present disclosure canbe significant, on the same order of magnitude as the axial forcecomponent, and is sufficient to overcome the fluid drag force at linearvelocities of up to 1 cm/s.

The lateral force can be increased by driving the transducer in higherorder mode shapes, as opposed to a form of vibration where the crystaleffectively moves as a piston having a uniform displacement. Theacoustic pressure is proportional to the driving voltage of thetransducer. The electrical power is proportional to the square of thevoltage. The transducer is typically a thin piezoelectric plate, withelectric field in the z-axis and primary displacement in the z-axis. Thetransducer is typically coupled on one side by air (i.e. the air gapwithin the transducer) and on the other side by the fluid of the cellculture media. The types of waves generated in the plate are known ascomposite waves. A subset of composite waves in the piezoelectric plateis similar to leaky symmetric (also referred to as compressional orextensional) Lamb waves. The piezoelectric nature of the plate typicallyresults in the excitation of symmetric Lamb waves. The waves are leakybecause they radiate into the water layer, which result in thegeneration of the acoustic standing waves in the water layer. Lamb wavesexist in thin plates of infinite extent with stress free conditions onits surfaces. Because the transducers of this embodiment are finite innature, the actual modal displacements are more complicated.

FIG. 12 shows the typical variation of the in-plane displacement(x-displacement) and out-of-plane displacement (y-displacement) acrossthe thickness of the plate, the in-plane displacement being an evenfunction across the thickness of the plate and the out-of-planedisplacement being an odd function. Because of the finite size of theplate, the displacement components vary across the width and length ofthe plate. In general, a (m,n) mode is a displacement mode of thetransducer in which there are m undulations in transducer displacementin the width direction and n undulations in the length direction, andwith the thickness variation as described in FIG. 14. The maximum numberof m and n is a function of the dimension of the crystal and thefrequency of excitation.

The transducers are driven so that the piezoelectric crystal vibrates inhigher order modes of the general formula (m, n), where m and n areindependently 1 or greater. Generally, the transducers will vibrate inhigher order modes than (2,2). Higher order modes will produce morenodes and antinodes, result in three-dimensional standing waves in thewater layer, characterized by strong gradients in the acoustic field inall directions, not only in the direction of the standing waves, butalso in the lateral directions. As a consequence, the acoustic gradientsresult in stronger trapping forces in the lateral direction. Put anotherway, driving the transducers to generate multi-modal vibrations cangenerate multiple standing waves from one piezoelectric crystal.

In embodiments, the pulsed voltage signal driving the transducer canhave a sinusoidal, square, sawtooth, or triangle waveform; and have afrequency of 500 kHz to 10 MHz. The pulsed voltage signal can be drivenwith pulse width modulation, which produces any desired waveform. Thepulsed voltage signal can also have amplitude or frequency modulationstart/stop capability to eliminate streaming. Again, the transducer isusually operated so that the acoustic standing wave remains onresonance. A feedback system is generally present for this purpose.

The transducer(s) is/are used to create a pressure field that generatesforces of the same order of magnitude both orthogonal to the standingwave direction and in the standing wave direction. When the forces areroughly the same order of magnitude, particles of size 0.1 microns to300 microns will be moved more effectively towards regions ofagglomeration (“trapping lines”). Because of the equally large gradientsin the orthogonal acoustophoretic force component, there are “hot spots”or particle collection regions that are not located in the regularlocations in the standing wave direction between the transducer and thereflector. Hot spots are located in the maxima or minima of acousticradiation potential. Such hot spots represent particle collectionlocations which allow for better wave transmission between thetransducer and the reflector during collection and strongerinter-particle forces, leading to faster and better particleagglomeration.

FIG. 13 and FIG. 14 are exploded views showing the various parts of areaction vessel that uses acoustophoresis to hold cells in place in agrowth volume. FIG. 15 provides only one chamber for one growth volume,while FIG. 16 has two chambers and can have two different growthvolumes.

Referring to FIG. 13, fluid enters the reaction vessel 190 through afour-port inlet 191. A transition piece 192 is provided to create plugflow through the chamber 193. A transducer 40 and a reflector 194 arelocated on opposite walls of the chamber for holding the cell culture inplace. Fluid then exits the chamber 193 and the reaction vessel throughoutlet 195. The growth volume is located in chamber 193.

FIG. 14 has two chambers 193. A system coupler 196 is placed between thetwo chambers 193 to join them together. A growth volume can be locatedin each chamber 193.

In biological applications, it is contemplated that all of the parts ofthe system (e.g. the reaction vessel, tubing leading to and from thebioreactor, the temperature-regulating jacket, etc.) can be separatedfrom each other and be disposable. Avoiding centrifuges and filtersallows better separation of the CHO cells without lowering the viabilityof the cells. The frequency of the transducers may also be varied toobtain optimal effectiveness for a given power.

The present disclosure has been described with reference to exemplaryembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the present disclosure be construed asincluding all such modifications and alterations insofar as they comewithin the scope of the appended claims or the equivalents thereof.

1. A process for collecting biomolecules from a cell culture,comprising: suspending the cell culture in a growth volume of thebioreactor, the bioreactor including at least one ultrasonic transducerand a reflector located opposite the at least one ultrasonic transducer,the at least one ultrasonic transducer being driven to produce amulti-dimensional acoustic standing wave that holds the cell culture inthe growth volume; and flowing a nutrient fluid stream through the cellculture to collect the biomolecules.
 2. The process of claim 1, whereinthe bioreactor further comprises a secondary filtering system locatedbetween the growth volume and a bioreactor outlet.
 3. The process ofclaim 2, further comprising activating the secondary filtering system ifthe multi-dimensional acoustic standing wave fails.
 4. The process ofclaim 1, wherein the multi-dimensional acoustic standing wave is inresonance.
 5. The process of claim 1, wherein the bioreactor where theat least one ultrasonic transducer is an array of elements.
 6. Theprocess of claim 1, wherein each ultrasonic transducer produces aplurality of multi-dimensional acoustic standing waves.
 7. The processof claim 1, wherein the bioreactor does not include an impeller withinthe growth volume.
 8. The process of claim 1, wherein the cell cultureis composed of Chinese hamster ovary (CHO) cells.
 9. The process ofclaim 1, wherein the biomolecules are monoclonal antibodies orrecombinant proteins.
 10. The process of claim 1, wherein themulti-dimensional acoustic standing wave has an axial force componentand a lateral force component which are of the same order of magnitude.11. The process of claim 1, wherein the ultrasonic transducer comprisesa piezoelectric material that can vibrate in a higher order mode shape.12. The process of claim 11, wherein the piezoelectric material has arectangular shape.
 13. The process of claim 1, wherein the ultrasonictransducer comprises: a housing having a top end, a bottom end, and aninterior volume; and a crystal at the bottom end of the housing havingan exposed exterior surface and an interior surface, the crystal beingable to vibrate when driven by a voltage signal.
 14. The process ofclaim 13, wherein a backing layer contacts the interior surface of thecrystal, the backing layer being made of a substantially acousticallytransparent material.
 15. The process of claim 14, wherein thesubstantially acoustically transparent material is balsa wood, cork, orfoam.
 16. The process of claim 14, wherein the substantiallyacoustically transparent material has a thickness of up to 1 inch. 17.The process of claim 14, wherein the substantially acousticallytransparent material is in the form of a lattice.
 18. The process ofclaim 13, wherein an exterior surface of the crystal is covered by awear surface material with a thickness of a half wavelength or less, thewear surface material being a urethane, epoxy, or silicone coating, orbeing made of aluminum oxide.
 19. The process of claim 13, wherein thecrystal has no backing layer or wear layer.
 20. The process of claim 1,wherein the multi-dimensional acoustic standing wave is athree-dimensional standing wave.